OBJECTIVES: To evaluate the relation between single nucleotide polymorphisms (SNPs) of tropoelastin gene and aortic dissection (AD) via identifying SNPs in the tropoelastin gene, and to detect the level of tropoelastin mRNA, elastin and elastic fibers. METHODS: The specimens of the AD group ( RESULTS: Seven SNP loci of the tropoelastin gene were detected in these samples. Among them, 5 SNP loci were polymorphic. The frequency of 3 SNP loci[rs2071307 (G/A), rs34945509 (C/T) and rs17855988 (G/C)] was significantly different between the AD group and the control group (all CONCLUSIONS The polymorphisms of rs2071307 (G/A), rs34945509 (C/T), and rs17855988(G/C) in the tropoelastin gene may eventually affect the synthesis of elastic fibers and they may play an important role in the occurrence of AD.
Aneurysm, Dissecting/genetics* ; Elastic Tissue ; Elastin/genetics* ; Humans ; Polymorphism, Single Nucleotide ; Tropoelastin/genetics*

Extracellular elastase-like protease is one of the key virulence proteases of Scedosporium aurantiacum. To date, little is known about this enzyme in terms of genetic information, structure, properties and virulence mechanism due to the difficulties in purification caused by its low secretion amount, high specific activity, uncompleted genome sequencing and annotation. This work investigated the gene, structure and enzymatic properties of this enzyme. The S. aurantiacum elastase-like protease from the fungal culture supernatant was analyzed through tandem mass spectrometry (MS/MS) approach, illustrating its primary structure. Bioinformatics tools were employed to predict the conserved domain and tertiary structure, the enzymatic properties were also studied. It turned out that S. aurantiacum extracellular elastase-like protease demonstrated well hydrolysis towards elastin and bovine achilles tendon collagen, with V_max of 18.14 μg/s and 17.57 μg/s respectively, better than fish scale gelatin, with the lowest hydrolysis effect on casein. Its activity towards elastin was lower than that of the elastase from porcine pancreas, with values of K_cat/K_m of 3.541 (μg/s) and 4.091 (μg/s), respectively. It was an alkaline protease, with optimal pH 8.2 and temperature 37 ^oC. Zn²⁺ promoted the enzymatic activity while Ca²⁺, Mg²⁺, Na⁺, elastatinal and PMSF inhibited its activity. Its sequence was similar to Paecilomyces lilacinus secreted serine protease (PDB Entry: c3f7oB_) with multiple conserved fractions each containing more than 7 amino acids, thus suitable for design of PCR primer. This study increased our knowledge on S. aurantiacum extracellular elastase-like protease in terms of structure and enzymatic properties, and may facilitate later studies on protein expression and virulence mechanism.
Animals ; Cattle ; Pancreatic Elastase/genetics* ; Elastin/genetics* ; Tandem Mass Spectrometry ; Serine Proteases/genetics*

OBJECTIVE: To carry out genetic diagnosis for a pedigree affected with cutis laxa. METHODS: Genomic DNA was extracted from peripheral blood samples from members of the pedigree and 50 unrelated healthy controls. Potential mutation was screened by next-generation sequencing and verified by Sanger sequencing. RESULTS: A heterozygous c.1985delG mutation was identified in the ELN gene among all patients from this pedigree. The same mutation was not found among unaffected family members and 50 healthy controls. CONCLUSION The genetic etiology for the pedigree has been elucidated, which has enabled genetic counseling and guidance for reproduction.
Cutis Laxa ; genetics ; Elastin ; genetics ; Heterozygote ; High-Throughput Nucleotide Sequencing ; Humans ; Mutation ; Pedigree

Antimicrobial peptides are the most promising alternatives to antibiotics. However, the strategy of producing antimicrobial peptides by recombinant technology is complicated and expensive, which is not conducive to the large-scale production. Oxysterlin 1 is a novel type of cecropin antimicrobial peptide mainly targeting on Gram-negative bacteria and is of low cytotoxicity. In this study, a simple and cost-effective method was developed to produce Oxysterlin 1 in Escherichia coli. The Oxysterlin 1 gene was cloned into a plasmid containing elastin-like polypeptide (ELP) and protein splicing elements (intein) to construct the recombinant expression plasmid (pET-ELP-I-Oxysterlin 1). The recombinant protein was mainly expressed in soluble form in E. coli, and then the target peptide can be purified with a simple salting out method followed by pH changing. The final yield of Oxysterlin 1 was about 1.2 mg/L, and the subsequent antimicrobial experiment showed the expected antimicrobial activity. This study holds promise for large-scale production of antimicrobial peptides and the in-depth study of its antimicrobial mechanism.
Elastin ; Escherichia coli/genetics* ; Inteins ; Peptides/pharmacology* ; Pore Forming Cytotoxic Proteins ; Recombinant Fusion Proteins/genetics*

Elastin-like polypeptides (ELPs) are temperature sensitive biopolymers composed of a Val-Pro-Gly-Xaa-Gly pentapeptide repeat that derived from a structural motif found in mammalian elastin. It was a promising tag for recombinant protein purification. Here, we de novo designed a novel ELPs gene and cloned it into the modified expression vector pET-22b(+). Then, we transformed the recombinant expression vector pET-22b-ELPs into Escherichia coli BL21(DE3). Upon induction by Isopropyl beta-D-Thiogalactoside (IPTG), ELPs was expressed and purified by a non-chromatographic purification method named inverse temperature cycling. The influences of salts types and concentrations on ELPs were also determined. The results showed that the transition temperature of the [KV8F-20] decreased to 19 degrees C by 0.4 mmol/L Na2CO3. Due to its small molecular weight and sensitivity to salt, the ELPs might be a useful purification tag, which can provide a reliable and simple non-chromatographic method for purification of the recombinant protein by inverse transition cycling.
Chromatography ; Elastin ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Peptides ; genetics ; isolation & purification ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Sodium Chloride ; pharmacology

BACKGROUNDThe pathological characteristics of abdominal aortic aneurysm (AAA) involved the regression of extracellular matrix (ECM) in aortic walls, especially elastic structure in medial layer. As the major structural protein of aorta, elastin contributes to the extensibility and elastic recoil of the vessels. We hypothesized that overexpression of elastin in vessel walls might regenerate the elastic structure of ECM, restore the elastic structure of the aneurysmal wall, and eventually lead to a reduction of aortic diameters (ADs) in an experimental model of AAA.

METHODSTropoelastin (TE) of Sprague Dawley (SD) rat was synthesized by reverse transcription polymerase chain reaction and used to construct adneviral vectors containing elastin precursor protein (AdTE-GFP). Cultured vascular smooth muscle cells (VSMCs) from aortas of male SD rats were transfected with AdTE-GFP, AdGFP, adenoviral vector (AdNull), and phosphate buffered saline (PBS). Immunofluorescence staining was performed to determine the expression of elastin in transfected cells. The expression of elastic fibers in ECM of VSMCs transfected with AdTE-GFP were detected by fluorescence microscopy and transmission electron microscopy (TEM) at 1, 3, and 5 days following gene transfer. The AAA vessel walls were infused with AdTE-GFP or an empty AdNull, or PBS directly into the aneurysmal lumen. ADs of the aneurysms were compared in infused aortas. Formation of new elastic fibers in vivo was assessed by hematoxylin and eosin, and elastic von-Giesson staining. Recombinant elastin-GFP in vivo was identified by immunohistochemical staining.

RESULTSElastic fibers were increased both in ECM of VSMC and in vessel walls after gene transfer. Histological studies revealed that the AdTE-GFP-transduced aortas had elastic fiber regeneration in the aneurysmal walls. The AdTE-GFP-transduced aortas showed a decreased AD (23.04% ± 14.49%, P < 0.01) in AAA vessel walls.

CONCLUSIONSElastic fibers have been successfully overexpressed both in vitro and in a rat model of AAA by a technique of gene transfer. The overexpression of elastic fibers within the aneurysmal tissue appeared to reverse the aneurysm dilatation in this model.

Animals ; Aortic Aneurysm, Abdominal ; metabolism ; therapy ; Elastic Tissue ; metabolism ; Elastin ; genetics ; metabolism ; Fluorescent Antibody Technique ; Male ; Muscle, Smooth, Vascular ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tropoelastin ; genetics ; metabolism

This study was conducted to examine the effectiveness of a gene transfer of human TGF beta 1 gene into smooth muscle cells and whether the TGF beta 1 can increase elastin expression of smooth muscle cells. With the help of DOTAP, smooth muscle cells were transfected with pMAMneoTGF beta 1. The positive cell clones were selected with G418. The stable transfection and expression of TGF beta 1 in the smooth muscle cells were determined by immunofluorescence analysis. The expression of elastin in the transfected and untransfected cells were determined by in situ hybridization. The adhesion force between smooth muscle cells and matrix was detected by micropipette system. The results showed abundant TGF beta 1 stable expression in smooth muscle cells. TGF beta 1 gene can increase two-three times elastin expression and increase the adhesion between smooth muscle cells and matrix. TGF beta 1 can be used in vascular tissue engineering to increase smooth muscle cells adhesion.
Cell Adhesion ; Cells, Cultured ; Elastin ; biosynthesis ; Humans ; In Situ Hybridization ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Transfection ; Transforming Growth Factor beta ; biosynthesis ; genetics ; physiology ; Transforming Growth Factor beta1